Dolors Costa

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Non-coding recurrent mutations in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3′ region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.

Cytogenetic risk stratification in chronic myelomonocytic leukemia

Background The prognostic value of cytogenetic findings in chronic myelomonocytic leukemia is unclear. Our purpose was to evaluate the independent prognostic impact of cytogenetic abnormalities in a large series of patients with chronic myelomonocytic leukemia included in the database of the Spanish Registry of Myelodysplastic Syndromes. Design and Methods We studied 414 patients with chronic myelomonocytic leukemia according to WHO criteria and with a successful conventional cytogenetic analysis at diagnosis. Different patient and disease characteristics were examined by univariate and multivariate methods to establish their relationship with overall survival and evolution to acute myeloid leukemia. Results Patients with abnormal karyotype (110 patients, 27%) had poorer overall survival (P=0.001) and higher risk of acute myeloid leukemia evolution (P=0.010). Based on outcome analysis, three cytogenetic risk categories were identified: low risk (normal karyotype or loss of Y chromosome as a single anomaly), high risk (presence of trisomy 8 or abnormalities of chromosome 7, or complex karyotype), and intermediate risk (all other abnormalities). Overall survival at five years for patients in the low, intermediate, and high risk cytogenetic categories was 35%, 26%, and 4%, respectively (P<0.001). Multivariate analysis confirmed that this new CMML-specific cytogenetic risk stratification was an independent prognostic variable for overall survival (P=0.001). Additionally, patients belonging to the high-risk cytogenetic category also had a higher risk of acute myeloid leukemia evolution on univariate (P=0.001) but not multivariate analysis. Conclusions Cytogenetic findings have a strong prognostic impact in patients with chronic myelomonocytic leukemia.

Gene Expression Profiling of Acute Myeloid Leukemia with Translocation t(8;16)(p11;p13) and MYST3-CREBBP Rearrangement Reveals a Distinctive Signature with a Specific Pattern of HOX Gene Expression

Acute myeloid leukemia (AML) with translocation t(8;16)(p11;p13) is an infrequent leukemia subtype with characteristic clinicobiological features. This translocation leads to fusion of MYST3 (MOZ) and CREBBP (CBP) genes, probably resulting in a disturbed transcriptional program of a myelomonocytic precursor. Nonetheless, its gene expression profile is unknown. We have analyzed the gene expression profile of 23 AML patients, including three with molecularly confirmed MYST3-CREBBP fusion gene, using oligonucleotide U133A arrays (Affymetrix). MYST3-CREBBP cases clustered together and clearly differentiated from samples with PML-RARalpha, RUNX1-RUNX1T1, and CBFbeta-MYH11 rearrangements. The relative expression of 46 genes, selected according to their differential expression in the high-density array study, was analyzed by low-density arrays in an additional series of 40 patients, which included 7 MYST3-CREBBP AML cases. Thus, genes such as prolactin (PRL) and proto-oncogene RET were confirmed to be specifically overexpressed in MYST3-CREBBP samples whereas genes such as CCND2, STAT5A, and STAT5B were differentially underexpressed in this AML category. Interestingly, MYST3-CREBBP AML exhibited a characteristic pattern of HOX expression, with up-regulation of HOXA9, HOXA10, and cofactor MEIS1 and marked down-regulation of other homeobox genes. This profile, with overexpression of FLT3, HOXA9, MEIS1, AKR7A2, CHD3, and APBA2, partially resembles that of AML with MLL rearrangement. In summary, this study shows the distinctive gene expression profile of MYST3-CREBBP AML, with overexpression of RET and PRL and a specific pattern of HOX gene expression.

Type I MOZ/CBP (MYST3/CREBBP) is the most common chimeric transcript in acute myeloid leukemia with t(8;16)(p11;p13) translocation

The t(8;16)(p11;p13) fuses the MOZ (MYST3) gene at 8p11 with CBP (CREBBP) at 16p13 and is associated with an infrequent but well‐defined type of acute myeloid leukemia (AML) that has unique morphocytochemical findings (monocytoid blast morphology with erythrophagocytosis and simultaneously positive for myeloperoxidase and nonspecific esterases). RT‐PCR amplification of MOZ/CBP (MYST3/CREBBP) chimera has proved difficult, with four different transcripts found in four reported cases. We studied 7 AML‐t(8;16) patients, 5 with cytogenetically demonstrated t(8;16) and 2 with similar morphocytochemical and immunophenotypical characteristics. Clinically, 3 cases presented as therapy‐related leukemia. Extramedullar involvement was observed at presentation in 2 patients and coagulopathy in 4. The clinicobiological findings confirmed the distinctiveness of this entity. Of note is the erythrophagocytosis in 5 of 7 cases and the immunological negativity for CD34 and CD117 and positivity for CD56. Using a new RT‐PCR strategy, we were able to amplify a specific band of 212 bp in six cases in which sequence analysis confirmed the presence of the previously described MOZ/CBP fusion transcript type I. This is the largest molecularly studied AML‐t(8;16) series, which demonstrates that MOZ/CBP breakpoints are usually clustered in intron 16 of MOZ and intron 2 of CBP. The newly designed single‐round PCR provides a simple tool for the molecular confirmation of MOZ/CBP rearrangement.

Incidence and Prognostic Impact of Secondary Cytogenetic Aberrations in a Series of 145 Patients with Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a mature B‐cell neoplasm with an aggressive behavior, characterized by the t(11;14)(q13;q32). Several secondary genetic abnormalities with a potential role in the oncogenic process have been described. Studies of large MCL series using conventional cytogenetics, and correlating with proliferation and survival, are scarce. We selected 145 MCL cases at diagnosis, displaying an aberrant karyotype, from centers belonging to the Spanish Cooperative Group for Hematological Cytogenetics. Histological subtype, proliferative index and survival data were ascertained. Combined cytogenetic and molecular analyses detected CCND1 translocations in all cases, mostly t(11;14)(q13;q32). Secondary aberrations were present in 58% of patients, the most frequent being deletions of 1p, 13q and 17p, 10p alterations and 3q gains. The most recurrent breakpoints were identified at 1p31‐32, 1p21‐22, 17p13, and 1p36. Aggressive blastoid/pleomorphic variants displayed a higher karyotypic complexity, a higher frequency of 1p and 17p deletions and 10p alterations, a higher proliferation index and poor survival. Gains of 3q and 13q and 17p13 losses were associated with reduced survival times. Interestingly, gains of 3q and 17p losses added prognostic significance to the morphology in a multivariate analysis. Our findings confirm previous observations indicating that proliferation index, morphology and several secondary genetic alterations (3q gains and 13q and 17p losses) have prognostic value in patients with MCL. Additionally, we observed that 3q gains and 17p losses detected by conventional cytogenetics are proliferation‐independent prognostic markers indicating poor outcome.

Different distribution of NOTCH1 mutations in chronic lymphocytic leukemia with isolated trisomy 12 or associated with other chromosomal alterations.

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in Western countries. Chromosomal abnormalities commonly found using conventional cytogenetics and FISH are del(11)(q22‐23), trisomy 12, del(13)(q14), and del(17)(p13). Trisomy 12 is the most frequent numerical abnormality in CLL. It can appear isolated or associated with other chromosomal aberrations, including t(14;18)(q32;q21) and trisomy 18. The aim of this study was to determine whether CLL patients with isolated trisomy 12 or associated with other chromosomal alterations have different clinico‐pathological features, including a different distribution NOTCH1 mutation. Patients were classified into four groups: Group 1, isolated trisomy 12 (n = 14); Group 2, trisomy 12 plus trisomy 18 (n = 4); Group 3, trisomy 12 plus t(14;18) (n = 8); and Group 4: patients with trisomy 12 plus other abnormalities not involving BCL2 (n = 28). The Binet stage and expression of ZAP70 were significantly different among cytogenetic groups. NOTCH1 mutations were detected in 6/12 (50%) patients from Group 1, 4/25 (16%) patients from Group 4, and in no patient from groups 2 and 3 (P = 0.020). Patients in Group 2 had a more rapid disease progression (median Treatment‐free Survival 2 months) as against patients from Groups 1 (50 months), 3 (69 months), or 4 (68 months; P = 0.001). These findings indicate that the distribution of NOTCH1 mutations in CLL with trisomy 12 is heterogeneous and that the presence of additional chromosomal abnormalities such as trisomy 18 could change the prognosis of these patients.

Response to lenalidomide in myelodysplastic syndromes with del(5q): influence of cytogenetics and mutations

Lenalidomide is an effective drug in low‐risk myelodysplastic syndromes (MDS) with isolated del(5q), although not all patients respond. Studies have suggested a role for TP53 mutations and karyotype complexity in disease progression and outcome. In order to assess the impact of complex karyotypes on treatment response and disease progression in 52 lenalidomide‐treated patients with del(5q) MDS, conventional G‐banding cytogenetics (CC), single nucleotide polymorphism array (SNP‐A), and genomic sequencing methods were used. SNP‐A analysis (with control sample, lymphocytes CD3+, in 30 cases) revealed 5q losses in all cases. Other recurrent abnormalities were infrequent and were not associated with lenalidomide responsiveness. Low karyotype complexity (by CC) and a high baseline platelet count (>280 × 109/l) were associated with the achievement of haematological response (P = 0·020, P = 0·013 respectively). Unmutated TP53 status showed a tendency for haematological response (P = 0·061). Complete cytogenetic response was not observed in any of the mutated TP53 cases. By multivariate analysis, the most important predictor for lenalidomide treatment failure was a platelet count <280 × 109/l (Odds Ratio = 6·17, P = 0·040). This study reveals the importance of a low baseline platelet count, karyotypic complexity and TP53 mutational status for response to lenalidomide treatment. It supports the molecular study of TP53 in MDS patients treated with lenalidomide.

Correlation between genetic polymorphisms of the hOCT1 and MDR1 genes and the response to imatinib in patients newly diagnosed with chronic-phase chronic myeloid leukemia

The association between seven polymorphisms in the genes hOCT1 and MDR1, encoding for imatinib transporter proteins, and the response to imatinib 400mg/daily was investigated in 65 patients newly diagnosed with chronic-phase chronic myeloid leukemia. The AA genotype at the rs6935207 hOCT1 polymorphic locus was not detected in patients with inadequate response to imatinib. The CC genotype at the rs1045642 (C3435T) MDR1 locus was associated with primary failure, whereas a T allele at the rs2032582 (G2677T/A) MDR1 locus seemed to protect from primary failure. Beside, the MDR1 haplotype 1236T-2677G-3435C was more frequently found in patients primarily resistant to imatinib.

CRITERIA FOR FETAL NUCHAL THICKNESS CUT-OFF: A RE-EVALUATION

An attempt has been made to establish a more effective cut‐off criterion for nuchal thickness (NT) and to assess the optimal gestational period for the prediction of trisomies 21 and 18. Reference intervals were established for NT from the tenth to the 18th week, using either gestation‐specific centiles or the parametric method. The measurements in 47 consecutive trisomy 21 and 18 trisomy 18 cases were plotted against these intervals. Assaying different cut‐off criteria for both the centile and the parametric methods, sensitivities and false‐positive rates for each gestational week were calculated and then compared with the commonly applied ‘two‐stepped’ cut‐off method (3 mm early, 6 mm later). The parametric method, based on a progressive rise, with +2·5 SD for the corresponding gestational week as a cut‐off value, showed the best performance (likelihood ratio 38) in the prediction for trisomy 21. The optimal gestational age was the 12–18 week period, with an overall sensitivity of 62 per cent (23/37) for an average false‐positive rate of 0·7 per cent. For trisomy 18, the most effective cut‐off was also +2·5 SD, and 10–13 gestational weeks as the optimal period, achieving 86 per cent (6/7) sensitivity for a 1·9 per cent false‐positive rate.